A natural single nucleotide mutation in the small regulatory RNA ArcZ of Dickeya solani switches off the antimicrobial activities against yeast and bacteria

The necrotrophic plant pathogenic bacterium Dickeya solani emerged in the potato agrosystem in Europe. All isolated strains of D. solani contain several large polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene clusters. Analogy with genes described in other bacteria suggests that the clusters ooc and zms are involved in the production of secondary metabolites of the oocydin and zeamine families, respectively. A third cluster named sol was recently shown to produce an antifungal molecule. In this study, we constructed mutants impaired in each of the three secondary metabolite clusters sol, ooc, and zms to compare first the phenotype of the D. solani wild-type strain D s0432-1 with its associated mutants. We demonstrated the antimicrobial functions of these three PKS/NRPS clusters against bacteria, yeasts or fungi. The cluster sol, conserved in several other Dickeya species, produces a secondary metabolite inhibiting yeasts. Phenotyping and comparative genomics of different D. solani wild-type isolates revealed that the small regulatory RNA ArcZ plays a major role in the control of the clusters sol and zms. A single-point mutation, conserved in some Dickeya wild-type strains, including the D. solani type strain IPO 2222, impairs the ArcZ function by affecting its processing into an active form.

to inhibit the growth of different yeast species. We had previously named this cluster ssm (for solani secondary metabolite) [19], and it was later renamed sol [14]. Finally, the comparative analysis of the genomes and phenotypes of several wild-type D. solani strains revealed the fundamental role of the small RNA ArcZ. This sRNA plays a major role in the regulation of the sol and zms clusters, which can be disrupted by a mutation in the arcZ sequence, conserved in some natural isolates.

Description and distribution of the three selected PKS/NRPS secondary metabolite clusters of D. solani
The gene clusters encoding complex NRPS and PKS involved in the production of secondary metabolites were named sol, ooc and zms, respectively ( Fig 1A). Their distribution in Dickeya species is summarized in Fig 1B (see also S1 Table for a complete list of the Dickeya strains with the indication of the presence or absence of each cluster).
The *42-kbp cluster sol contains the 12 genes solABCDEFGHIJKL (Fig 1A). It is widely conserved in the genus Dickeya, i.e., in all sequenced D. solani, D. aquatica, D. fangzhongdai, D. poaceiphila and D. zeae genomes, in some D. dadantii strains (such as NCPPB 898, NCPPB 3537, DSM18020, but not the model strain 3937), in some D. undicola strains (FVG1, FVG10), and in some D. oryzae strains (such as S20 and FVG03). The structure of the metabolite produced from this cluster has not been elucidated yet but the function of this cluster has been described by us in our 2021 preprint article [19] and others in 2022 [14]. The sol cluster produces solanimycin that targets fungi and yeast of the Ascomycota phylum.
The *80-kbp cluster ooc ( Fig 1A) is highly similar to the oocBCDEFGJKLMNOPQR-STUVW cluster of S. plymuthica A153. It is present in all the sequenced genomes of D. solani and D. dianthicola, in some D. oryzae strains (such as ZYY5, EC1, DZ2Q and ZJU1202) and in a few other species. In S. plymuthica A153, disruption of this gene cluster abolished bioactivity against the ascomycete Verticillium dahliae and the oomycetes Pythium ultimum. This cluster produces oocydin A [15], a chlorinated macrolide, powerfully active against plant pathogenic oomycetes [20]. Since various D. solani strains inhibit V. dahliae and P. ultimum growth [16], it was suggested, on the basis of gene sequence homologies and similar cluster organization, that D. solani also produces oocydin A.
The 55-kbp cluster zms encodes mixed fatty acid synthase FAS/PKS and hybrid NRPS/PKS enzymes ( Fig 1A). Its genomic organization is identical to the D. oryzae EC1 zeamine cluster and related to the S. plymuthica AS12 zeamine cluster [17,21]. While the zmsABCDEF-GIJKLMNPQRS cluster directing zeamine biosynthesis is present only in a few D. oryzae strains, it is conserved in all the sequenced D. solani and D. fangzhongdai genomes, which suggest secondary acquisition by horizontal gene transfer in D. oryzae [13,21]. After reclassification of several D. zeae strains in the novel species D. oryzae [22], the zms cluster appeared to be absent in the genomes of true D. zeae strains. It is present in some, but not all, D. oryzae rice strains (ZYY5, EC1, DZ2Q and ZJU1202) and in only one D. dadantii strain (A622-S1-A17). The zeamine biosynthetic clusters from D. oryzae EC1 and D. solani Ds0432-1 share from 59 to 94% identity at individual protein level [21]. Zeamine-related antibiotics are polyaminoamide molecules toxic to a wide range of pro-and eukaryotic organisms such as bacteria, fungi, oomycetes, plants, and nematodes [23]. Mutation of the zeamine synthase gene zmsA in D. oryzae EC1 attenuates the inhibition of rice seed germination [17] and suppresses antibacterial activity against E. coli [17]. Zeamine produced by S. plymuthica kills nematodes and ascomycete yeast [24]. D. solani IPO 2222 can also kill the nematode Caenorhabditis elegans but not as quickly as S. plymuthica [24].
To interrupt the synthesis of the secondary molecules produced by these three clusters, we constructed from a Nal R Gm R derivative of WT D. solani D s0432-1 in-frame deletion mutants inactivating a PKS or NRPS gene of each cluster, which is predicted to interrupt metabolite synthesis. The inhibitory effects of the mutants ΔsolG, ΔoocL and ΔzmsA against fungi, bacteria and yeasts were compared with that of the parental strain. To further analyze possible synergistic effects, double mutants ΔsolGΔoocL, ΔsolGΔzmsA and ΔoocLΔzmsA as well as the triple mutant (hereafter called Δ3) were also constructed.

The D. solani oocydin cluster ooc inhibits growth of phytopathogenic ascomycete fungi, and the sol cluster is also involved in the inhibition of some fungi
We compared the ability of the WT D. solani strain D s0432-1 and its derived mutants to inhibit the growth of Botrytis cinerea, Magnaporthe oryzae and Sclerotinia sclerotiorum, three fungi eukaryotes of the phylum Ascomycota. The center of a potato dextrose agar (PDA) plate was inoculated with fungal mycelium, and 5 μl of overnight bacterial culture of each D. solani strain grown in M63 minimal medium with sucrose was deposited at the periphery of the plate (Fig 2). The different D. solani strains showed similar overall growth in liquid culture (S1 Fig). After incubation at 25˚C for several days, we observed a growth inhibition of the three fungi by the D. solani WT and the ΔsolG and ΔzmsA mutants. In contrast, the ΔoocL mutant seemed less effective: B. cinerea and M. oryzae mycelium growth was completely unaffected, while a small zone of inhibition was observed for S. sclerotiorum (Fig 2). This suggests that the ΔoocL mutant may also produce another molecule that inhibits this fungus. The phenotypes were fully restored in the reverted strain ΔoocL/oocL + . Double and triple mutants of the secondary metabolite clusters containing the oocL deletion showed no differences from the ΔoocL mutant with B. cinerea and M. oryzae. However, the ΔoocLΔsolG double mutant and the Δ3 triple mutant were completely unable to inhibit S. sclerotiorum growth (Fig 2). These data suggest that, in our assay on PDA plate, the ooc cluster is mainly responsible for the majority of ascomycete inhibition and that the sol cluster is secondary involved in the inhibition of some fungi. In contrast, the zms cluster does not seem to be involved in fungal inhibition.

The D. solani zeamine cluster inhibits bacterial growth
The zeamine cluster of D. oryzae EC1 is responsible for a bactericidal activity against Escherichia coli DH5α [17]. D. oryzae EC1 and D. solani zeamine biosynthetic genes share a high degree of similarity [21]. We thus evaluated the capacity of the D. solani strain D s0432-1 and its derived mutants to inhibit the growth of Gram-positive and Gram-negative bacteria (Fig 3). Only the ΔzmsA mutant was unable to inhibit Bacillus subtilis growth, indicating that WT D. solani produces an active zeamine antibiotic (Fig 3). The WT phenotype was restored in the reverted ΔzmsA/zmsA+ strain. We also tested the ability of D. solani to inhibit growth of the other Gram-positive bacteria Streptomyces scabiei, a plant pathogen causing the potato disease common scab [25]. An inhibition of S. scabiei growth was observed, except with the ΔzmsA mutant. No phenotype difference was observed between the single ΔzmsA mutant and the double and Genes are indicated using the NCBI nomenclature of the NCBI reference genome sequence NZ_CP017453.1 of D. solani D s0432-1. XXXXX is the digital number in BJD21_RSXXXXX, which corresponds to the locus tag. Arrowheads show gene orientations. Color code indicates gene function. The red-framed arrows indicate genes targeted for in-frame deletion performed in this study. (B) MLSA tree positioning strains within the Dickeya genus. The evolutionary history was inferred by using the Maximum Likelihood method. The presence (green frame) or absence (red frame) of the cluster sol, ooc, and zms are indicated in front of each strain. The distribution of these clusters in 155 Dickeya strains whose genome has been sequenced is given in S1 Table. https://doi.org/10.1371/journal.pgen.1010725.g001 triple mutants containing the zmsA deletion, suggesting that, out of the three clusters, only zms is required for Gram-positive bacterial growth inhibition. We also tested the ability of D. solani to inhibit growth of the Gram-negative bacteria E. coli, D. dadantii and Pectobacterium atrosepticum (Fig 3). E. coli DH5α was, like B. subtilis and S. scabiei, inhibited by the WT strain through zms activity, albeit much less potently (Figs 3 and S2). No inhibition of the two other pectinolytic bacteria D. dadantii and P. atrosepticum growth was observed. In conclusion, out of the

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three clusters analyzed, only the zeamine-like molecule potentially produced by the zms cluster has an antibacterial activity against some Gram-positive and Gram-negative bacteria.

The D. solani clusters sol and zms inhibits ascomycete yeast growth
Since zeamine produced by S. plymuthica A153 has previously been shown to be toxic to the ascomycete yeast Saccharomyces cerevisiae [24], we tested the capacity of D. solani D s0432-1 and its derivative mutant to inhibit the growth of the yeasts S. cerevisiae, Kluyveromyces lactis, a predominant eukaryote during cheese productions [26], and Candida albicans, an opportunistic human pathogen. In a growth inhibition assay performed in Yeast Peptone Dextrose (YPD) solid agar medium, we observed a strong inhibition of K. lactis by WT D. solani, a lower inhibition of S. cerevisiae, and a slight inhibition of C. albicans (Fig 4). The ΔsolG mutant was almost completely deficient in its ability to inhibit yeast growth, while the ΔsolG/solG + revertant strain showed a restored WT phenotype. The cluster sol is thus primarily responsible for the antieukaryotic action against these yeasts on YPD (Fig 4). Nevertheless, a weak halo of inhibition of the yeasts K. lactis and S. cerevisiae was observable around the mutant ΔsolG. This halo disappeared around the double mutant ΔsolGΔzmsA and the triple mutant Δ3, suggesting that the zms cluster is also implicated in yeast inhibition, but at a lower level than that observed for the sol cluster. In conclusion, although the zeamine cluster of D. solani D s0432-1 is involved in the inhibition of yeast growth, as shown for S. plymuthica A153 [24], D. solani D s0432-1 also possesses the cluster sol that produces another antiyeast molecule.

The WT D. solani strains IPO 2222 and IFB0223 are unable to inhibit yeast and bacterial growth
We showed that D. solani D s0432-1 has the capacity to inhibit the growth of a wide range of microorganisms, including filamentous fungi, bacteria, and yeasts. We therefore tested whether other WT D. solani strains isolated from the environment in Europe, either from lesions of potato tubers or plants, or from potato rhizosphere, possess this ability. We focused our work on D. solani strains available in national strain collections and whose genome sequence is available. These strains are D s0432-1, IFB0099, IFB0158, IFB0223, IFB0484, IPO 3337, IPO 3494, IPO 3793 and the D. solani type strain IPO 2222. We tested their capacity to inhibit the bacterium B. subtilis, the yeast K. lactis and the fungus S. sclerotiorum ( Fig 5A). All strains were able to inhibit the growth of S. sclerotiorum. The inhibition of B. subtilis and K. lactis was clearly observed for all the strains except IPO 2222 and IFB0223. These strong phenotypic differences between IPO 2222 and IFB0223 versus the other WT D. solani strains could not be explained by any nucleotide variabilities in the sol, zms, and ooc clusters. All the selected strains carry the sol, zms, and ooc clusters, with 100% conservation at the nucleotide level (including in the promoter regions). Nonetheless, there are certain changes at the genome level, most notably some nucleotide variability with the presence of SNPs (single-nucleotide The radius of each inhibition zone was measured. A statistical difference was significant only between the WT and the Δsol mutants (Mann-Whitney test; * p-value < 0.05). For C. albicans, the inhibition zone was too small to be quantified. All experiments were performed in 4 replicates.
https://doi.org/10.1371/journal.pgen.1010725.g004 polymorphism) and InDel (insertion or deletion), outside the clusters sol, zms, and ooc. Hence, we searched for variations in the whole genomes by comparing two groups: strains IFB0223 and IPO 2222 that are defective for the growth inhibition of yeast and bacteria, versus strains D s0432-1, IPO 3337 and IFB0099 that are efficient for these inhibitions (S2 Table).
A SNP was observed with an identical nucleotide in the strains D s0432-1, IPO3337 and IFB0099 (a G at position 90 of arcZ) but different nucleotide in the strain IPO2222 (a A at position 90 of arcZ) (position 2530087 according to IPO 2222 genome). This variation is positioned in the 3' region of the small RNA arcZ, known to be a key area interacting with targeted mRNA for post-translational regulation in other bacterial species (Fig 5C). The ArcZ variants of D s0432-1 and IPO 2222 will be referred to as ArcZ 1 and ArcZ 2 respectively in the remainder of this article. In the strain IFB0223, we observed a 3-kbp chromosomal deletion covering the gene arcZ, and extending between the genes arcB and osmY ( Fig 5B). In other D. solani strains, this region includes arcZ, elbB, mtgA, and a gene encoding an alginate lyase domain (pfam05426). These discoveries led us to hypothesize that the inability of strains IFB0223 and IPO 2222 to inhibit yeast and bacterial growth could be due to an ArcZ deficiency.
Before going further, we ordered the D. solani IPO 2222 type strain stored in the BCCM collection (LMG 25993) to analyze its genotype and phenotype. Using PCR amplification and Sanger sequencing of arcZ, we confirmed that the arcZ allele of LMG 25993 is arcZ 2 , similarly to the IPO 2222 strain used in our experiments. Our laboratory strain IPO 2222 and LMG 25993 have the same inhibition phenotype, i.e., an inability to inhibit yeast and bacterial growth (S3 Fig). The arcZ 1 allele is required for zms and sol antimicrobial activities sRNAs are posttranscriptional regulators that most commonly influence gene translation by base-pairing to target mRNAs. In E. coli, this regulation often requires the RNA chaperones Hfq or ProQ [27][28][29]. ArcZ belongs to what is called the "core sRNAs" because it is highly conserved among enterobacterial species [30]. In order to determine if ArcZ is required for secondary metabolite production, the ΔarcZ mutants of D. solani strains D s0432-1 and IPO 2222 were constructed, and their phenotypes compared to those of the WT strains ( Fig 6A). In M63 sucrose, the mutants grew as well as the parental WT strains (S4 Fig). In our inhibition assay against B. subtilis and K. lactis, no inhibition zones were observed around the ΔarcZ 1 mutant of D s0432-1 (Fig 6A), demonstrating that ArcZ 1 is required for the synthesis of the metabolites produced from the sol and zms clusters in D s0432-1. However, the ΔarcZ 1 mutant of D. solani D s0432-1 was still able to inhibit the growth of the fungus S. sclerotiorum, although a slight decrease in the antifungal effect was observed. Thus, ArcZ does not appear to be fully required for ooc cluster expression, which is consistent with the results obtained with D. solani IFB0223, the naturally arcZ deletion mutant capable of inhibiting S. sclerotiorum growth (Figs 5A and 6A). D. solani IPO 2222 WT inhibited neither bacteria nor yeast but inhibited the growth of fungi, and the phenotype of the IPO 2222 ΔarcZ 2 mutant was similar to that of the IPO 2222 WT or the D s0432-1 ΔarcZ 1 mutant. This suggests that ArcZ 2 might be inactive.
The sol and zms clusters' expression is promoted by the arcZ 1 allele, but not arcZ 2 To further demonstrate that ArcZ 1 controls the sol and zms clusters, we constructed transcriptional fusions between the promoter region of the sol, zms, and ooc clusters, and the luxCDABE operon. We introduced sol::luxCDABE, zms::luxCDABE and ooc::luxCDABE into WT D s0431-1, WT IPO 2222, WT IFB0223, and the mutants ΔarcZ 1 D s0432-1 and ΔarcZ 2 IPO 2222. Luminescence of the strains were measured (Fig 6B). We detected a strong transcriptional activity of the sol cluster in WT D s0432-1, while its activity was 229-fold lower in the ΔarcZ 1 background. WT IPO 2222, its ΔarcZ 2 mutant, and IFB0223 all exhibited low sol transcriptional activity similar to the ΔarcZ 1 D s0432-1 mutant. Regarding the transcriptional activity of the cluster zms, WT D s0432-1 had a significant *3-fold stronger transcriptional activity than the ΔarcZ 1 D s0432-1 mutant, WT IPO 2222, and its ΔarcZ 2 mutant. Surprisingly, the ΔarcZ 1 D s0432-1 mutant, WT IPO 2222, and its ΔarcZ 2 mutant had a stronger zms transcriptional activity compared to IFB0223, suggesting the existence of other genomic differences between IPO 2222 and IFB0223 that could modulate zms transcriptional activity. Finally, a slight 1.8-fold decrease of ooc transcriptional activity was measured between WT D s0432-1 and its ΔarcZ 1 D s0432-1 mutant. These findings indicate that ArcZ has a modest effect on ooc transcriptional activity while promoting the expression of zms and sol clusters.
We next looked for predictions of ArcZ 1 interactions with mRNA targets in the -200/+100 region around the start codon (+1) of each D. solani D s0432-1 gene using the CopraRNA algorithm [31] (S3 Table). No putative interactions were predicted around the start codons of the first genes of each cluster (solA, zmsA and oocJ). This result, together with transcriptional fusion analyses, suggests that ArcZ may not interact directly with the 5' ends of the sol and zms mRNAs but rather indirectly by regulating the expression of a transcriptional factor that regulates these clusters at the transcriptional level.

The G90A mutation in arcZ 2 of D. solani IPO 2222 causes a loss of function of zms and sol clusters
To verify the effect of the G90A mutation in arcZ, we performed a heterologous complementation experiment by transferring the allele arcZ 1 from D s0432-1 or arcZ 2 from IPO 2222 in D. solani WT strains D s0432-1 or IPO 2222 or their ΔarcZ derivatives. The alleles arcZ 1 and arcZ 2 were also transferred into D. solani IFB0223 naturally deleted for arcZ.
The two allelic forms arcZ 1 and arcZ 2 were cloned with their own promoter in the plasmid pWSK29-oriT, a mobilizable low copy plasmid with pSC101 origin and ampicillin resistance gene. DNA sequencing confirmed that the only difference between the two plasmids is the G90A mutation in pWSK29-oriT-arcZ 2 . The recombinant plasmids and the empty vector were transferred by conjugation into WT or ΔarcZ D. solani D s0432-1 or IPO 2222 strains, as well as WT D. solani IFB0223 (naturally deleted for arcZ). The phenotypes of the conjugants were determined by testing their antimicrobial activity against B. subtilis, K. lactis and S. sclerotiorum ( Fig 6C).
The transfer of arcZ 1 in ΔarcZ 1 Ds0432-1 restored the WT phenotypes, proving that the allele arcZ 1 activates the antimicrobial activities of the sol and zms clusters, while the antifungal activity was unaffected. In contrast, the transfer of arcZ 2 in the ΔarcZ 1 DS0432-1 strain did not restore the WT phenotypes. Thus, the difference in complementation between arcZ 1 and arcZ 2 is due to the presence of the G90A mutation that impairs ArcZ 2 function.
An identical result was obtained with the ΔarcZ 2 mutant of IPO 2222 or the IFB0223 strain carrying the pWSK29-oriT, pWSK29-oriT-arcZ 1 and pWSK29-oriT-arcZ 2 . Indeed, the transfer of arcZ 1 in strains WT IPO 2222, ΔarcZ 2 IPO 2222 or IFB0223 activates the antimicrobial activities of the sol and zms clusters. In contrast, the transfer of arcZ 2 in these strains did not. To gain further insight into the consequence of the G90A mutation in arcZ, Northern-blot experiments were performed to detect the sRNAs ArcZ 1 in WT D s0432-1 and ArcZ 2 in WT IPO 2222 (Fig 7). A probe specific to the 3' part of ArcZ 1 and ArcZ 2 was used (annealing from nucleotide 91 to 132). An abundant smaller transcript (~60nt) representing the processed 3 0 fragment of ArcZ 1 was detected in WT D s0432-1 but not in the mutant D s0432-1 ΔarcZ 1 (Fig  7, compared lanes 1 and 2). The full-length form corresponding to the ArcZ 1 precursor was barely detected. This confirms that the processed form of ArcZ 1 is predominant, like observed in E. coli, Salmonella, and Photorhabdus [30,32,33]. In IPO 2222, only the full-length ArcZ 2 was detected in the WT strain whereas no ArcZ 2 transcript was detected in the IPO 2222 ΔarcZ 2 mutant (Fig 7, compared lanes 5 and 6). This result suggests that the G90A mutation present in the arcZ 2 allele of IPO 2222 prevents ArcZ 2 processing in a shorter functional transcript.
Next, we realized Northern-blot with the D. solani ΔarcZ strains expressing either the arcZ 1 or arcZ 2 allele from the plasmid pWSK29-oriT-arcZ. On one hand, Northern blot showed that D s0432-1ΔarcZ 1 , IPO 2222 ΔarcZ 2 and IFB0223 (natural ΔarcZ) expressing arcZ 1 from D s0432-1 accumulate the processed short transcript of ArcZ 1 (Fig 7, lanes 3, 7 and 10) and have the ability to inhibit yeast and bacteria (Fig 6). On the other hand, D s0432-1ΔarcZ 1 , IPO 2222 ΔarcZ 2 and IFB0223 expressing arcZ 2 from IPO 2222 showed the accumulation of the nonprocessed transcript of ArcZ 2 (Fig 7, lanes 3, 7 and 10) and an inability to inhibit yeast and bacteria (Fig 6). These results support the hypothesis that the G90A mutation in ArcZ alters the processing of the ArcZ precursor in a shorter form, leading to its inability to play a correct regulatory function on the sol and zms clusters.

Discussion
PKS/NRPS secondary metabolite pathways are a great source of molecules with antieukaryotic or antibacterial activity, giving the bacteria that synthesize them a competitive advantage over other organisms. Targeted mutagenesis of the D. solani D s0432-1 chromosome has allowed us to specifically study the involvement of three secondary metabolite biosynthesis clusters encoded by all the D. solani strains for which genome sequence is available. We focused our work on strain D s0432-1, one of the most virulent D. solani strains [9]. We have demonstrated that D. solani D s0432-1 is able to inhibit the growth of a variety of living microorganisms. We tested bio-activities against Gram-negative bacteria (E. coli, P. atrosepticum, D. dadantii), Gram-positive bacteria (B. subtilis, S. scabiei), ascomycete yeasts (S. cerevisiae, K. lactis, C. albicans), and ascomycete fungi (B. cinerea, M. oryzae and S. sclerotiorum). The cluster ooc encodes a biosynthesis pathway that produces an oocydin-like molecule, a chlorinated macrocyclic lactone molecule having antifungal, antioomycete and antitumor activities in S. plymuthica [15,16]. It was previously shown that the D. solani strains MK10, MK16 and IPO 2222, all encoding the oocydin cluster, inhibit the growth of the ascomycete fungus V. dahliae and the oomycete P. ultimum [16]. Our study showed that D. solani D s0432-1 prevents the growth of the ascomycete fungi B. cinerea, M. oryzae and S. sclerotiorum, and that inactivation of the oocL gene suppressed the inhibition of the three fungi without any visible effect on the bacteria or yeasts tested. We can thus conclude that the ooc cluster of D. solani produces an antiascomycete molecule.
By testing other mutants of the other secondary metabolite clusters, we showed that the mutant ΔsolG was nearly completely unable to prevent the growth of the ascomycete yeast S. cerevisiae and K. lactis. Therefore, the sol cluster appears to be responsible for yeast inhibition.

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D. solani D s0432-1 also inhibits the human pathogen yeast C. albicans, but its inhibitory activity appears to be weaker than that observed with S. cerevisiae and K. lactis in our inhibition assay. This observation on the sol cluster paves the way for the identification of a new molecule with highly specific inhibitory activity against yeasts. The sol cluster is conserved in several Dickeya genomes but rarely in other bacterial genera, except in some Rouxiella species. The structure of the molecule and its target remains to be elucidated.
Our study also showed that D. solani D s0432-1 has an antibacterial activity linked to the cluster zms, which encodes a zeamine biosynthetic pathway. Zeamine produced by S. plymuthica kills B. subtilis, the yeasts S. cerevisiae and Schizosaccharomyces pombe, and the nematode Caenorhabditis elegans [24]. We showed that D. solani D s0432-1, but not the ΔzmsA mutant, can clearly inhibit the growth of two Gram-positive bacteria, B. subtilis and S. scabiei. Since D. solani and S. scabiei are two potato pathogens, they might be in competition for the same ecological niche. We also observed a slight inhibition exerted by D. solani D s0432-1 against E. coli, but no inhibition towards D. dadantii and P. atrosepticum. Zeamine resistance of D. dadantii could be explained by the presence in its genome of the desAB genes encoding an RND pump identified in D. oryzae EC1 and involved in zeamine efflux [34]. In-frame deletion of desA or desB in D. oryzae EC1 leads to a zeamine sensitive phenotype [34]. In in planta Tn-seq experiments with D. dadantii 3937 [35], mutants of the genes desA and desB (Dda3937_00787 and Dda3937_00786 respectively) did not display a significant negative or positive variation (Log2 fold-changes -0.09 and +0.44, respectively), suggesting that this RND efflux pump does not play a significant role during D. dadantii chicory infection, contrary to the D. dadantii RND efflux pump AcrAB that appeared to be essential for virulence [35]. While P. atrosepticum is not inhibited by D. solani D s0432-1, it does not have the desAB genes. Zeamine resistance could be provided by another efflux pump or by a different mechanism in P. atrosepticum. It is worth remembering that the D. solani D s0432-1 ΔzmsA mutant grows slightly faster than the WT strain after reaching OD 600nm 0.4 (S1 Fig). This small difference in growth might be explained by the lack of need of the mutant to adapt to the presence of zeamine in the medium. We also observed that D. solani D s0432-1 ΔsolG produced a very slight halo of inhibition against S. cerevisiae and K. lactis, barely visible in our tests. This weak halo disappears in the double mutant ΔsolGΔzmsA and the triple mutant ΔsolGΔoocLΔzmsA, showing that the zeamine produced by the D. solani zms cluster has also an anti-yeast action. It is important to note that zeamine produced by S. plymuthica A153 is bioactive against S. cerevisiae and S. pombe [24]. Thus, the presence of three clusters encoding secondary metabolites allows D. solani D s0432-1 to produce an arsenal of bioactive secondary metabolites against a variety of living microorganisms. The cluster sol produces an unknown molecule active against ascomycete yeasts, the cluster ooc produces an oocydin-like molecule active against ascomycete fungi and the cluster zms produces a zeamine-like molecule active against bacteria and ascomycete yeast. It is now clear that D. solani synthesizes several molecules to inhibit the growth of ascomycetes. Through a variety of regulatory mechanisms, the bacterium may always be able to express at least one of the sol, zms, and ooc clusters. This can make it particularly effective in the struggle against rival microorganisms. In future studies, it would be instructive to investigate D. solani behavior toward other organisms found in the environment, such as amoebas, nematodes, or even insects.
We then expanded our studies to a larger set of D. solani strains isolated in different European countries and years. Through the combination of phenotypic analysis and comparative genomics, confirmed by mutant construction, we demonstrated that the ArcZ sRNA plays a major role in activating the antimicrobial activities of the clusters sol and zms. We first observed that some D. solani WT strains are defective for yeast inhibition and possess different arcZ alleles, a deletion in the case of strain IFB0223, or the presence of the SNP G90A in the 3' part of arcZ in the type strain IPO 2222. We demonstrated that these arcZ alterations significantly impair the D. solani ability to inhibit other bacteria and yeasts. These arcZ-related genomic changes represent a minority of D. solani isolates, only two of the nine strains phenotypically tested. Hence, our results show that despite a probable clonal origin, environmental strains of D. solani can present very different characteristics due to a single mutation into a regulatory sRNA. The arcZ polymorphism may be one of the explanations for the variability of phenotypes and virulence observed between genetically close D. solani strains. A global investigation of the polymorphism of all the small RNAs within the genomes of the same species could give surprising results. This study shows that it is critical to pay attention to the allelic diversity due to SNPs present not only in protein coding genes but also in sRNA genes.
ArcZ is a well-known regulatory sRNA that is associated with the chaperone Hfq. The ArcZ primary transcript (121 to 134nt-long) is converted by the essential endoribonuclease RNAse E [36] to a shorter stable of about 60 nt RNA retaining the seed region. The precise length of this short processed RNAs depends on the bacterial species [30,33,37,38]. In E. coli, the processed short form of ArcZ promotes rpoS translation while suppressing the expression of several other genes. In anaerobic conditions, arcZ is repressed and translation of RpoS is low [37]. RpoS is the RNA polymerase alternative sigma factor that governs the general stress response, which is activated by a wide range of conditions. It controls 10% of the E. coli genome [39]. RpoS can control positively or negatively the production of metabolites such as antibiotics [16,40]. The rpoS mRNA is not the only ArcZ direct target. In Photorhabdus and Xenorhabdus, ArcZ pairs with the mRNA encoding HexA, a transcriptional repressor of the expression of specific metabolite gene clusters [33]. In Dickeya species, the regulator gene pecT is the orthologous gene to hexA. In D. dadantii 3937, the pecT mRNA is also a direct target of ArcZ. Short processed ArcZ binds to Hfq and basepairs with the 5'UTR region of pecT mRNA, repressing its translation [38]. PecT is, directly and indirectly, involved in the expression of genes encoding plant cell wall degrading enzymes and virulence factors [38]. In the phytopathogen Erwinia amylovora, ArcZ also participates in the positive control of T3SS, exopolysaccharide production, biofilm formation, and motility [41]. Finally, another known direct target of ArcZ is the flhD mRNA [42,43]. FlhD forms with FlhC the master regulator FlhDC that controls motility in bacteria [44]. Although we demonstrated the critical positive role of ArcZ in the regulation of sol and zms expression, and secondary metabolite production in D. solani, it remains to understand the precise mechanism of ArcZ action on the sol and zms clusters. Our CopraRNA analysis indicates no putative binding between the processed form of ArcZ 1 and the 5'UTR region of solA and zmsA. However, the pecT 5'UTR, but not flhD and rpoS 5'UTRs, is detected as a putative target of ArcZ 1 (S3 Table). It should be kept in mind that bioinformatics analyses remain predictions. For example, CopraRNA does not detect the interaction between E. coli ArcZ and the 5'UTR of the rpoS mRNA. Much work remains to be done to elucidate the mode of action of ArcZ in Dickeya solani.
Another point to consider is a more global role of D. solani ArcZ exerted on other cellular functions such as motility and virulence. ArcZ targets in D. solani need to be investigated in the future. It is worth pointing out that the G90 nucleotide is in the 3' region of ArcZ that interacts with the target mRNAs rpoS, flhD, or pecT in other bacteria (S5 Fig). Northern-blot analyses revealed that ArcZ 2 of D. solani IPO 2222 is not processed in the stable shorter sRNA as ArcZ 1 of D. solani D s0432-1. The G90A mutation in IPO 2222 ArcZ 2 is predicted to modify the secondary structure of the ArcZ precursor form (Fig 7B). Since maturation by RNase E is essential for target regulation by the ArcZ sRNA in Salmonella [36], we conclude that the absence of a short form of ArcZ in IPO 2222 is probably the main cause of the loss of regulatory function of the IPO 2222 ArcZ 2 .
The type strain D. solani IPO 2222 is largely used in academic laboratories as a model to study the D. solani species. In light of our findings, it could be important to compare data obtained with the strain IPO 2222 with those obtained with other D. solani strains. The arcZ mutations may be responsible, at least partially, for the lower virulence of IPO 2222 and IFB0223 observed previously in comparison to D s0432-1 [9]. ArcZ is expressed in aerobic condition in stationary phase. Thus, in IFB0223 and the type strain IPO 2222, one can assume that each gene controlled by ArcZ in this condition will not be properly regulated at the post-transcriptional level. IPO 2222 exhibited a low number of genes with oxygen-dependent transcription [45]. It might be possible that the missing functional ArcZ in this strain prevents the oxygen-dependent response of some genes. Besides, IPO 2222 was isolated from infected seed potatoes in the Netherlands in 2007 [46]. It is less virulent than D s0432-1 [9] but can still successfully colonize potato and bittersweet nightshade (Solanum dulcamara) plants [47,48]. These observations imply that ArcZ might not be necessary for a successful plant infection. However, when D. solani is present on the phylosphere, where oxygen is available, ArcZ might be crucial for outcompeting other microorganisms. It will be necessary to conduct more research to validate these hypotheses.
Even strains with the same arcZ, sol, ooc, and zms genomic sequences as D s0432-1, showed heterogeneity in their ability to inhibit other microorganisms. For example, IPO 3494 and IFB0484 inhibited B. subtilis better than other D. solani strains (Fig 5A). Other specific mutations in the genomes of these strains, which could also modify the activity of secondary metabolite clusters, could explain these phenotypic variations.
We first described the inhibitory function of the sol cluster of the D. solani strain D s0432-1 against the yeasts S. cerevisiae and K. lactis in a 2021 bioRxiv preprint [19]. Very recently, in November 2022, Matilla et al. published results on the sol cluster of the D. solani strain MK10 [14]. The authors described the antifungal activity of the uncharacterized molecule that they called solanimycin [14]. Matilla et al. also reported the inhibition observed with the D. solani strain IPO 2222 against Verticillium dahliae and S. pombe ([14], S1 Fig of the article). In our conditions, strain IPO 2222 was able to inhibit the fungus S. sclerotiorum but not the yeast K. lactis (Fig 5). In comparison to our study, Matilla et al used different fungal species but also different growth conditions (medium, temperature, exposure time, etc). By random mutagenesis of strain MK10, they identified RsmA and the two Dickeya quorum-sensing systems ExpI/R and Vfm as regulators of the sol cluster [14]. Interestingly, the Rsm regulatory pathway of D. dadantii 3937 is regulated by PecT, which is under the ArcZ control [38]. Our CopraRNA analysis identified pecT, and vfmX, one of the Vfm quorum sensing genes, as putative targets of ArcZ, but not rsmA, expI and expR, (S3 Table).
Further studies are now necessary to specify the biological targets of solanimycin and to clarify the role in sol expression of the sRNA ArcZ, quorum-sensing Vfm and Exp, and transcriptional regulators PecT or RsmA, in order to better understand the regulatory network controlling the production of secondary metabolites in D. solani. We are currently investigating the regulatory pathway of secondary metabolite clusters involving ArcZ.
In conclusion, our results provide the basis for further investigation of a novel metabolite capable of limiting yeast growth and, at the same time, open doors to understand how the ArcZ sRNA acts as a global regulator to regulate two different clusters of secondary metabolites in D. solani. It also shows how a single mutation in a small RNA can greatly modify the phenotype of bacterial wild-type strains that are very close phylogenetically.

Growth inhibition assay of bacteria and yeast
D. solani strains were grown for 24 h at 30˚C with shaking in M63 medium supplemented with 1% sucrose. B. subtilis, E. coli, D. dadantii and P. atrosepticum were grown in LB at 30˚C with shaking overnight. The next day, the OD 600 of the cultures of D. solani were adjusted to 2. The temperature of melted LB agar was lowered to around 40˚C (just before the agar resolidified). 100 mL of the LB agar in surfusion were mixed with 100 μl of the OD 600 1 culture of B. subtilis, E. coli, D. dadantii or P. atrosepticum. 30 mL of inoculated LB agar were poured in 12-by 12-cm square plates. Then 5 μl of the OD 600 2 cultures of D. solani were spotted onto the inoculated square plates which were incubated at 30˚C for 24-48 h before visualization of the inhibition zone.
The same experiment was conducted with S. scabiei, except that it was grown for 3 days in TSB at 28˚C and TSB agar was poured in the square plates. With the yeasts S. cerevisiae, K. lactis, and C. albicans, a similar protocol was used with YPD medium and with OD 600 2 culture of yeast per 30mL of medium.

Growth inhibition of fungal strains
S. sclerotiorum, B. cinerea and M. oryzae were grown onto PDA plates at 25˚C for 5, 7 and 10 days respectively. D. solani strains were grown for 24 h at 30˚C with shaking in M63 medium supplemented with 1% sucrose. The OD 600 of these overnight cultures were adjusted to 2. Then, 5 μl of the bacterial suspensions were spotted onto PDA plates with 5 mm agar plugs of fungus at the center of the Petri dish. The radius of fungus inhibition zone was measured.

Construction of the MLSA tree positioning strains within the Dickeya genus
Details on the methods used for the construction of MLSA tree positioning strains are given in S1 Supplementary Methods.

Constructions of the mutant strains and plasmids
All the material and methods for the construction of the strains and plasmids used in this study are given in S1 Supplementary Methods.

RNA isolation and Northern detection
All the material and methods for RNA isolation and Northern detection performed in this study are given in S1 Supplementary Methods. Supporting information S1 Fig. Growth curves of D. solani strains and mutant derivatives. A 96-well plate containing M63 medium with 1% sucrose was inoculated with the strains used in this study at an OD of 0.06. The growth of each strain was determined by measuring OD 600 every 20 minutes during 2 days in a TECAN device. The different strains showed similar overall growth, except for the light grey points where a low but significant fitness gain was observed for the mutants